Bottom Line:
We found that YB-1 interacts directly with TOPO1 (but not with TOPO2) and promotes TOPO1 catalytic activity.Furthermore, we found that the interaction is prevented by pretreatment with the antioxidant agent, N-acetyl cysteine, and that YB-1 downregulation renders cells resistant to CPT.Our findings suggest that nuclear YB-1 serves as an intracellular promoter of TOPO1 catalytic activity that enhances CPT sensitivity through its direct interaction with TOPO1.

Background: The Y-box binding protein 1 (YB-1) possesses pleiotropic functions through its interactions with various cellular proteins, and its high expression levels make it a potential useful prognostic biomarker for cancer cells. Eukaryotic DNA topoisomerases, such as DNA topoisomerase 1 (TOPO1) and DNA topoisomerase 2 (TOPO2), are the essential DNA metabolism regulators that usually overexpressed in cancer cells, and multiple proteins have been reported to regulate the enzyme activity and the clinical efficacy of their inhibitors. The present study unraveled the interaction of YB-1 with TOPO1, and further investigated the related function and potential mechanisms during the interaction.

Methods: The direct association of TOPO1 with specific domain of YB-1 was explored by co-immunoprecipitation and GST pull-down assays. The interaction function was further clarified by DNA relaxation assays, co-immunoprecipitation and WST-8 assays with in vitro gain- and loss- of function models.

Results: We found that YB-1 interacts directly with TOPO1 (but not with TOPO2) and promotes TOPO1 catalytic activity. Interactions between YB-1 and TOPO1 increased when cancer cells were treated with the TOPO1 inhibitor, camptothecin (CPT), but not with the TOPO2 inhibitor, adriamycin (ADM). Furthermore, we found that the interaction is prevented by pretreatment with the antioxidant agent, N-acetyl cysteine, and that YB-1 downregulation renders cells resistant to CPT.

Conclusions: Our findings suggest that nuclear YB-1 serves as an intracellular promoter of TOPO1 catalytic activity that enhances CPT sensitivity through its direct interaction with TOPO1.

Fig1: YB-1 binds to TOPO1. A. Endogenous YB-1 binds to endogenous TOPO1 in human gastric cancer cells. Nuclear extracts (NE) from HGC-27 cells were subjected to immunoprecipitation with anti-TOPO1 or anti-TOPO2 antibodies, or a goat-IgG antibody control. The immunoprecipitates and the input from NE and cytosolic extracts (CE) were subjected to western blot analysis for TOPO2 (upper panel), TOPO1 (middle panel) and YB-1 (lower panel). B. Endogenous YB-1 binds to endogenous TOPO1 in human pancreatic cancer cells. Nuclear extracts (NE) from PANC-1 cells was subjected to immunoprecipitation with a TOPO1 antibody, TOPO2 antibody, or a goat-IgG antibody control. The immunoprecipitates and input were subjected to western blot analysis for TOPO2 (upper panel), TOPO1 (middle panel) and YB-1 (lower panel). C. Endogenous YB-1 binds to endogenous TOPO1 in human prostate cancer cells. Nuclear extracts (NE) from PC-3 cells was subjected to immunoprecipitation with a TOPO1 antibody, or a goat-IgG antibody control. The immunoprecipitates and the input from NE and cytosolic extracts (CE) were subjected to western blot analysis for TOPO1 (upper panel), and YB-1 (lower panel). D. TOPO1 in cell extracts binds to GST-YB-1 protein. Purified GST or GST-YB-1 protein was incubated, respectively, with 500 μg of cytosolic extracts (CE) and nuclear extracts (NE) from PC-3 cells for 4 h at 4°C. Proteins pulled down with the bait (which was immobilized on glutathione beads) were separated by SDS-PAGE. And then two parallel gels were subjected to western blot and Coomassie blue staining, respectively. Proteins transferred to the membrane were probed with an anti-TOPO1 antibody. The band labeled by an asterisk was identified as TOPO1.

Mentions:
To determine whether TOPO1 interacted with YB-1 in mammalian cells, we performed co-immunoprecipitation experiment. In human gastric cancer HGC-27 (Figure 1A) and pancreatic cancer PANC-1 cells (Figure 1B), TOPO2 and TOPO1 were observed in the nuclear extracts and immunoprecipitates with the TOPO2 and TOPO1 antibody, respectively (upper and middle panel). In the co-immunoprecipitation assays (lower panel), YB-1 was observed in both nuclear and cytosolic extracts. YB-1 was also found to interact with TOPO1, but not TOPO2 or goat IgG. Endogenous YB-1-TOPO1 interaction was further confirmed in human prostate cancer PC-3 cells (Figure 1C), but not in human lung cancer A549 and cervical cancer HeLa cells (data not shown). To establish whether this interaction was also observed in vitro, we performed GST pull-down assays, respectively, using cytosolic extracts and nuclear extracts from PC-3 cells, with either GST-YB-1 or GST protein as the bait (Figure 1D). As anticipated, GST-YB-1, but not GST, bound TOPO1. To determine whether YB-1 interacted with TOPO1 directly, and to identify the TOPO1 binding region in YB-1, we performed pull-down assays using a recombinant ThioHis-TOPO1 fusion protein and GST fusion proteins containing either full-length YB-1 or its mutant derivatives, GST-YB-1 Δ1–Δ3 (Figure 2A). ThioHis-TOPO1 bound to GST-YB-1, GST-YB-1 Δ2, Δ3, but not to GST-YB-1 Δ1 (Figure 2B). With the nuclease-treated PC-3 nuclear extracts, we further confirmed the direct association of YB-1 and TOPO1 in the cells. As seen in Figure 2C, GST-YB-1, GST-YB-1 Δ2, Δ3, but not GST or GST-YB-1 Δ1, bound TOPO1. These findings indicate that YB-1 binds directly to TOPO1 in the nucleus of human cancer cells, and the binding sites were specified as the cold shock domain (CSD) and the C-terminal region of YB-1.Figure 1

Fig1: YB-1 binds to TOPO1. A. Endogenous YB-1 binds to endogenous TOPO1 in human gastric cancer cells. Nuclear extracts (NE) from HGC-27 cells were subjected to immunoprecipitation with anti-TOPO1 or anti-TOPO2 antibodies, or a goat-IgG antibody control. The immunoprecipitates and the input from NE and cytosolic extracts (CE) were subjected to western blot analysis for TOPO2 (upper panel), TOPO1 (middle panel) and YB-1 (lower panel). B. Endogenous YB-1 binds to endogenous TOPO1 in human pancreatic cancer cells. Nuclear extracts (NE) from PANC-1 cells was subjected to immunoprecipitation with a TOPO1 antibody, TOPO2 antibody, or a goat-IgG antibody control. The immunoprecipitates and input were subjected to western blot analysis for TOPO2 (upper panel), TOPO1 (middle panel) and YB-1 (lower panel). C. Endogenous YB-1 binds to endogenous TOPO1 in human prostate cancer cells. Nuclear extracts (NE) from PC-3 cells was subjected to immunoprecipitation with a TOPO1 antibody, or a goat-IgG antibody control. The immunoprecipitates and the input from NE and cytosolic extracts (CE) were subjected to western blot analysis for TOPO1 (upper panel), and YB-1 (lower panel). D. TOPO1 in cell extracts binds to GST-YB-1 protein. Purified GST or GST-YB-1 protein was incubated, respectively, with 500 μg of cytosolic extracts (CE) and nuclear extracts (NE) from PC-3 cells for 4 h at 4°C. Proteins pulled down with the bait (which was immobilized on glutathione beads) were separated by SDS-PAGE. And then two parallel gels were subjected to western blot and Coomassie blue staining, respectively. Proteins transferred to the membrane were probed with an anti-TOPO1 antibody. The band labeled by an asterisk was identified as TOPO1.

Mentions:
To determine whether TOPO1 interacted with YB-1 in mammalian cells, we performed co-immunoprecipitation experiment. In human gastric cancer HGC-27 (Figure 1A) and pancreatic cancer PANC-1 cells (Figure 1B), TOPO2 and TOPO1 were observed in the nuclear extracts and immunoprecipitates with the TOPO2 and TOPO1 antibody, respectively (upper and middle panel). In the co-immunoprecipitation assays (lower panel), YB-1 was observed in both nuclear and cytosolic extracts. YB-1 was also found to interact with TOPO1, but not TOPO2 or goat IgG. Endogenous YB-1-TOPO1 interaction was further confirmed in human prostate cancer PC-3 cells (Figure 1C), but not in human lung cancer A549 and cervical cancer HeLa cells (data not shown). To establish whether this interaction was also observed in vitro, we performed GST pull-down assays, respectively, using cytosolic extracts and nuclear extracts from PC-3 cells, with either GST-YB-1 or GST protein as the bait (Figure 1D). As anticipated, GST-YB-1, but not GST, bound TOPO1. To determine whether YB-1 interacted with TOPO1 directly, and to identify the TOPO1 binding region in YB-1, we performed pull-down assays using a recombinant ThioHis-TOPO1 fusion protein and GST fusion proteins containing either full-length YB-1 or its mutant derivatives, GST-YB-1 Δ1–Δ3 (Figure 2A). ThioHis-TOPO1 bound to GST-YB-1, GST-YB-1 Δ2, Δ3, but not to GST-YB-1 Δ1 (Figure 2B). With the nuclease-treated PC-3 nuclear extracts, we further confirmed the direct association of YB-1 and TOPO1 in the cells. As seen in Figure 2C, GST-YB-1, GST-YB-1 Δ2, Δ3, but not GST or GST-YB-1 Δ1, bound TOPO1. These findings indicate that YB-1 binds directly to TOPO1 in the nucleus of human cancer cells, and the binding sites were specified as the cold shock domain (CSD) and the C-terminal region of YB-1.Figure 1

Bottom Line:
We found that YB-1 interacts directly with TOPO1 (but not with TOPO2) and promotes TOPO1 catalytic activity.Furthermore, we found that the interaction is prevented by pretreatment with the antioxidant agent, N-acetyl cysteine, and that YB-1 downregulation renders cells resistant to CPT.Our findings suggest that nuclear YB-1 serves as an intracellular promoter of TOPO1 catalytic activity that enhances CPT sensitivity through its direct interaction with TOPO1.

Background: The Y-box binding protein 1 (YB-1) possesses pleiotropic functions through its interactions with various cellular proteins, and its high expression levels make it a potential useful prognostic biomarker for cancer cells. Eukaryotic DNA topoisomerases, such as DNA topoisomerase 1 (TOPO1) and DNA topoisomerase 2 (TOPO2), are the essential DNA metabolism regulators that usually overexpressed in cancer cells, and multiple proteins have been reported to regulate the enzyme activity and the clinical efficacy of their inhibitors. The present study unraveled the interaction of YB-1 with TOPO1, and further investigated the related function and potential mechanisms during the interaction.

Methods: The direct association of TOPO1 with specific domain of YB-1 was explored by co-immunoprecipitation and GST pull-down assays. The interaction function was further clarified by DNA relaxation assays, co-immunoprecipitation and WST-8 assays with in vitro gain- and loss- of function models.

Results: We found that YB-1 interacts directly with TOPO1 (but not with TOPO2) and promotes TOPO1 catalytic activity. Interactions between YB-1 and TOPO1 increased when cancer cells were treated with the TOPO1 inhibitor, camptothecin (CPT), but not with the TOPO2 inhibitor, adriamycin (ADM). Furthermore, we found that the interaction is prevented by pretreatment with the antioxidant agent, N-acetyl cysteine, and that YB-1 downregulation renders cells resistant to CPT.

Conclusions: Our findings suggest that nuclear YB-1 serves as an intracellular promoter of TOPO1 catalytic activity that enhances CPT sensitivity through its direct interaction with TOPO1.